Antibody Function and Structure

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Questions and Answers

Explain how antibodies contribute to the process of neutralizing foreign objects within the body.

Antibodies bind to antigens on the surface of foreign objects, marking them for destruction by immune cells or neutralizing their ability to infect cells.

Describe the general structure of an antibody molecule and explain how this structure enables its function.

Antibodies are Y-shaped molecules with two heavy chains and two light chains. The variable regions at the tips of the 'Y' provide antigen-binding specificity, while the constant region mediates effector functions.

If a patient's blood test reveals a high level of a specific type of antibody, what might this indicate about their current or past health status?

A high level of a specific antibody suggests either a current infection or a previous exposure to the corresponding antigen, indicating an active or past immune response.

Explain why antibodies are also known as immunoglobulins (Ig). Further, describe the characteristics of Immunoglobulin G (IgG).

Antibodies are known as immunoglobulins (Ig) because they are globular proteins involved in the immune system. Immunoglobulin G (IgG) is the most abundant antibody in the blood and provides long-term immunity. Signup and view all the answers

Describe the role of antibodies in identifying foreign objects.

Antibodies recognize and bind to specific antigens on the surface of foreign objects, marking them for immune responses. Signup and view all the answers

Explain the relationship between an antigen, an antigenic determinant (epitope), and an antibody. How do they interact?

An antigen is a substance that triggers an immune response, specifically the production of an antibody. An antigenic determinant, or epitope, is the specific part of the antigen that the antibody binds to. Signup and view all the answers

Briefly describe what is meant by the term 'immunoassay'.

An immunoassay is a laboratory technique that uses the binding between an antigen and its corresponding antibody to identify and measure the amount of a specific antigen or antibody in a sample. Signup and view all the answers

Explain what is meant by 'variable region' and 'constant region' in the context of an antibody structure and their roles.

The variable region of an antibody is the part that binds to a specific antigen. Conversely, the constant region determines the antibody's class and effector functions (e.g., complement activation). Signup and view all the answers

How do the immunoglobulin classes and subclasses differ across species such as mouse, rat, and humans?

While IgG and IgM are common classes across mouse, rat, and humans, the subclasses within IgG (e.g., IgG1, IgG2a) and other classes like IgA, IgD, and IgE are not uniformly present or categorized across these species. Signup and view all the answers

Briefly describe the initial step involved in generating specific antibodies within a host animal following antigen administration.

After an antigen is injected into the host, the animal's immune system recognizes it as foreign and initiates an immune response. B cells that recognize the antigen become activated. Signup and view all the answers

Consider a scenario where a researcher is developing an immunoassay. Why is it important to understand the specific immunoglobulin subclasses (e.g., IgG1, IgG2) in the species they are working with?

Understanding immunoglobulin subclasses is important because each subclass can have different effector functions and binding affinities. This knowledge helps in selecting the appropriate antibody for detection or capture in the immunoassay, optimizing its sensitivity and specificity. Signup and view all the answers

Explain why animals like mice, rats, or rabbits are preferred when only small quantities of antibodies are needed.

Smaller animals such as mice, rats, and rabbits are preferred when only small quantities of antibodies are needed due to their lower blood volume and ease of handling, which makes them more practical and cost-effective for small-scale antibody production. Signup and view all the answers

Outline two advantages of using larger animals like goats or horses for antibody production.

Larger animals like goats or horses allow for the production of large quantities of antibodies due to their greater blood volume and can sustain more frequent antibody harvesting compared to smaller animals. Signup and view all the answers

If an experiment requires the use of an antibody that can activate complement, which region of the antibody molecule would be most critical for this function, and why?

The constant region (Fc region) of the antibody is most critical for complement activation because it contains the binding site for complement proteins. This interaction triggers the complement cascade, leading to opsonization, inflammation, and cell lysis. Signup and view all the answers

In the context of antibody structure, what is the significance of the heavy and light chains?

Heavy and light chains are the two types of polypeptide chains that make up an antibody molecule. The heavy chains determine the antibody's class (e.g., IgG, IgM), while both heavy and light chains contribute to the antigen-binding site. Signup and view all the answers

What determines the specificity of the antibodies produced in response to antigen injection?

The specificity of the produced antibodies is determined by the unique structure of the injected antigen. The B-cells produce antibodies that specifically bind to that antigen's structure. Signup and view all the answers

How might prior exposure to an antigen (or a similar one) affect the antibody production process in a mammal?

Prior exposure to an antigen can result in a quicker and more robust antibody response upon subsequent exposure, due to immunological memory. Memory B cells will differentiate and produce antibodies faster. Signup and view all the answers

Explain why monoclonal antibodies are beneficial, compared to polyclonal antibodies, in research and diagnostic applications. Give an example.

Monoclonal antibodies offer high specificity because they are produced by a single clone of B cells and bind to a single epitope. An example of a beneficial use for it is in ELISA assays because of the increased levels of precision and accuracy. Signup and view all the answers

Explain why the selection of a suitable adjuvant is critical when injecting an antigen into an animal for antibody production.

Adjuvants enhance the immune response, leading to higher antibody titers. They work by activating immune cells and prolonging antigen exposure to the immune system. Signup and view all the answers

Describe one method for purifying antibodies from the serum of an immunized animal, and briefly explain its principle.

Affinity chromatography is commonly used. It involves using a column with an antigen or antibody-binding protein immobilized on it, allowing specific antibodies to bind while other serum proteins are washed away. The bound antibodies are then eluted. Signup and view all the answers

List two factors that should be considered when assessing the quality of antibodies produced in an animal model?

The factors include antibody titer (concentration) and specificity. Titer measures the amount of antibody produced in response to a given antigen, and specificity refers to how selectively the antibody binds to the intended antigen without cross-reactivity. Signup and view all the answers

Briefly describe the purpose of creating hybridoma cells in monoclonal antibody production.

Hybridoma cells are created to produce a continuous supply of a specific antibody. They are formed by fusing a B-lymphocyte, which produces the desired antibody, with a cancer cell, which provides immortality. Signup and view all the answers

Explain how a primary antibody differs from a secondary antibody in terms of antigen binding.

A primary antibody binds directly to a specific antigen, whereas a secondary antibody binds to the primary antibody instead of directly to the antigen. Signup and view all the answers

What is an epitope, and what role does it play in antibody binding?

An epitope is a specific site on an antigen that an antibody recognizes and binds to. It is the part of the antigen that directly interacts with the binding site of the antibody. Signup and view all the answers

In the context of antibody production, what is meant by the term 'monoclonal'?

Monoclonal refers to antibodies that are produced by a single clone of cells and are therefore identical in their structure and antigen specificity. Signup and view all the answers

Describe a scenario where using a secondary antibody would be beneficial, compared to using only a primary antibody.

Using a secondary antibody can amplify the signal in detection assays. Because multiple secondary antibodies can bind to a single primary antibody, this enhances the overall detection sensitivity. Signup and view all the answers

Explain the process of creating a hybridoma from start to finish.

First, an animal is exposed to an antigen. Plasma B cells are extracted from the animal, and fused with immortal myeloma (cancer) cells, creating hybridoma cells. These cells are then screened to identify and isolate the hybridomas that produce the desired antibody. Signup and view all the answers

Why is it important for monoclonal antibodies to target a specific epitope on an antigen?

Targeting a specific epitope ensures high specificity and reduces off-target binding. This precision is crucial for diagnostic and therapeutic applications where accurate targeting is essential. Signup and view all the answers

How might monoclonal antibodies be used in cancer treatment, based on their properties?

Monoclonal antibodies can be designed to specifically bind to cancer cells, either marking them for destruction by the immune system or delivering therapeutic agents directly to the tumor site. Signup and view all the answers

Why is alkaline phosphatase (ALP) often favored over peroxidase in certain enzyme-linked immunosorbent assays (ELISAs)?

Alkaline phosphatase is often favored due to its greater stability compared to peroxidase, despite being slightly more expensive and having a lower enzyme-to-antibody conjugation ratio. Signup and view all the answers

Explain why EDTA can be used as a stopping reagent for alkaline phosphatase reactions, and why acids can also be used.

EDTA stops ALP reactions by chelating the metal ions required for enzyme activity. Acids stop ALP by lowering the pH, which inactivates the enzyme. Signup and view all the answers

Describe how the presence of alcohol enhances the reaction rate of Beta-Galactosidase (BGAL).

Alcohols enhance the reaction rate of BGAL, making it suitable for assays performed on hydrophobic membrane surfaces. Signup and view all the answers

What are the products formed when Beta-Galactosidase (BGAL) hydrolyzes ONPG, and which product is responsible for the measurable color change?

BGAL hydrolyzes ONPG into galactose and ortho-nitrophenol. Ortho-nitrophenol produces the yellow color. Signup and view all the answers

Why is p-NPP not recommended for kinetic analysis?

pNPP is not recommended for kinetic analysis because its reaction must be stopped with 1N NaOH. Signup and view all the answers

How do chemiluminescent HRP substrates enable the detection of horseradish peroxidase (HRP)?

Chemiluminescent HRP substrates emit light as a result of the reaction with HRP, allowing for highly sensitive, nonradioactive detection. Signup and view all the answers

Compare and contrast the use of ONPG and pNPP as substrates in enzyme assays, noting the enzymes they are specific to and the methods used to stop their reactions.

ONPG is specific to Beta-Galactosidase and is stopped with 1M Sodium Carbonate, while pNPP is specific to Alkaline Phosphatase and is stopped with 1N NaOH. Signup and view all the answers

Describe how the molecular weight of an enzyme affects its use in assays.

The molecular weight of an enzyme can affect its conjugation ratio and ability to penetrate certain matrices or interact with other molecules. Smaller enzymes have a higher conjugation ratio. Signup and view all the answers

In a Sandwich ELISA, why is a polyclonal antibody often chosen as the capture antibody?

A polyclonal antibody is used as the capture antibody to maximize the amount of antigen that can be pulled down from the sample. Signup and view all the answers

What is the purpose of blocking non-specific binding sites with bovine serum albumin (BSA) in an ELISA?

Blocking with BSA prevents antibodies from binding to the microtiter plate itself, reducing background noise and improving the accuracy of the assay. Signup and view all the answers

Describe the role of the enzyme-linked secondary antibody in an ELISA.

The enzyme-linked secondary antibody binds to the primary antibody and helps to produce a detectable signal by acting on a substrate, enabling the quantification of the target antigen. Signup and view all the answers

In an ELISA, what is being measured when the substrate for the enzyme is introduced?

When the substrate is introduced, the amount of product formed by the enzyme's reaction is measured, which directly correlates with the quantity of the target antigen present in the sample. Signup and view all the answers

In an ELISA, what quality of the secondary antibody is important for signal amplification?

The enzyme label linked to the secondary antibody is important for signal amplification. Signup and view all the answers

Explain why washing steps are crucial in performing an ELISA.

Washing steps are crucial to remove any unbound antibodies or reagents, reducing background noise and ensuring that only specific binding events contribute to the final signal. Signup and view all the answers

Describe the difference between direct and indirect ELISA methods.

In direct ELISA, the primary antibody is directly labeled with an enzyme, whereas in indirect ELISA, the primary antibody is unlabeled and a labeled secondary antibody is used for detection. Signup and view all the answers

In the context of ELISA, explain the term 'antigen masking' and how it might affect the results.

Antigen masking occurs when the epitope recognized by the antibody is hidden or altered, preventing the antibody from binding and leading to a false negative result or underestimation of antigen concentration. Signup and view all the answers

Flashcards

What are antibodies?

Proteins in the blood that identify and neutralize foreign objects.

Antibody abbreviation

Ig. Gamma globulin proteins found in blood.